Through expression cloning we have isolated a cDNA-encoding SREBP cleavage-activating protein (SCAP), which regulates cholesterol metabolism by stimulating cleavage of transcription factors SREBP-1 and -2, thereby releasing them from membranes. The cDNA was isolated from Chinese hamster ovary cells with a dominant mutation that renders them resistant to sterol-mediated suppression of cholesterol synthesis and uptake. Sterol resistance was traced to a G-->A transition at codon 443 of SCAP, changing aspartic acid to asparagine. The D443N mutation enhances the cleavage-stimulating ability of SCAP and renders it resistant to inhibition by sterols. SCAP has multiple membrane-spanning regions, five of which resemble the sterol-sensing domain of HMG CoA reductase, an endoplasmic reticulum enzyme whose degradation is accelerated by sterols. SCAP appears to be a central regulator of cholesterol metabolism in animal cells.